Milk which is marketed in Canada and the United States must be fortified with vitamin A and D3 (1,2). Regulatory agencies have set standards specifying the minimum amount of vitamins A and D3 to be added to milk products. Fortified fluid milk products add value to the agricultural and dairy industry in that consumers seek products with essential vitamins and nutrients. Milk processors typically assert general claims of vitamin fortification levels because current methodology is too costly and time consuming to implement testing on a batch basis.
Vitamins A and D3 are potentially toxic to humans at higher concentrations. Since the margin between the nutritionally desirable intake of vitamins and harmful excess is small (3,4,5) it is important that errors in fortification levels be detectable in the shortest possible time. Currently available methodology for these analyses is laborious, tedious, and expensive, and adds to the high cost of production to dairy processors. Currently available methods for detecting vitamin A and D3 metabolites include binding assays, receptor proteins, high performance liquid chromatography (HPLC), and gas chromatography-mass spectrometry (GC-MS) (6,7,8,9). Such analyses in any laboratory are time consuming, require skill and expertise on the part of analysts, and require large capital investments for equipment.
Vitamins A and D3 are fat soluble and they are bound by fat molecules in a dairy product such as milk. Accordingly, they must be extracted in lengthy extraction steps. Since vitamins A and D are both labile to heat, light, and oxidation, laborious extraction results in loss of the vitamin in the preparative steps, and often requires 3 to 5 days for the completion of the analyses.
Vitamin D plays an active role in the homeostatic mechanism that controls the concentration of calcium ion in plasma. Vitamin D is transported to various sites in the body where it is activated. The activated forms of the vitamin act on the target tissues, thereby causing an increase in calcium content. The activation of vitamin D is regulated in a negative feedback system by plasma calcium. The most biologically active form of vitamin D is 1,25-dihydroxycholecalciferol or calcitriol, which is formed by two successive hydroxylations of vitamin D. That is, calcitriol is formed by the sequential hydroxylation of vitamin D at C-25 in the liver and at C-1 in the kidney. Various other analogs can be produced by hydroxylation at C-24 and C-26. The above mentioned biologically active forms of hydroxylated vitamin D are synthesized in the body only and are not found in dairy or agri-food products. Vitamin D3, or cholecalciferol, is the form used as an additive and is also produced in the body when the skin, which contains the provitamin 7-dehydrocholesterol, is exposed to sunlight.
Currently, the principal assay for Vitamin D3 in dairy samples which has been developed is a two step high pressure liquid chromatography (HPLC) assay, whereby the extracted vitamin in about 200 ml of the hexane is lyophilized to about 2 ml and the fraction corresponding to vitamin D is isolated by HPLC and collected manually. This collected fraction of vitamin D is again lyophilized, and dissolved in about 0.5 ml of methanol and loaded again on reverse phase HPLC. This assay employs an expensive and costly laboratory set up and requires trained and skilled personnel to handle advanced instrumentation like HPLC (8,9,10).
The principal assays for vitamin A include laborious extractions and the use of HPLC, or a direct extraction with the detection of vitamin A by spectrofluorometer. The latter process has limitations due to the interference from other compounds that has fluorescence at the same wavelength. Moreover, because vitamins A and D3 are sensitive to UV-light, they might lose some of their activity due to extensive extractions, purifications and storage conditions.
The standard assays, which employ one or two step (HPLC) are illustrated, for example, by references (6-9) and (10).
Therefore, there is a need in the art for methods and kits for quantifying vitamins A and D in a fluid sample, and dairy products in particular.